Design, synthesis and evaluation of novel tetrahydropyridothienopyrimidin-ureas as cytotoxic and anti-angiogenic agents

The novel derivatives of tetrahydropyridothienopyrimidine-based compounds have been designed and efficiently synthesized with good yields through seven steps reaction. The anticancer activity of compounds 11a-y has been evaluated against MCF-7, PC-3, HEPG-2, SW-480, and HUVEC cell lines by MTT assay. The target compounds showed IC50 values between 2.81–29.6 μg/mL and were compared with sorafenib as a reference drug. Among them, compound 11n showed high cytotoxic activity against four out of five examined cell lines and was 14 times more selective against MRC5. The flow cytometric analysis confirmed the induction of apoptotic cell death by this compound against HUVEC and MCF-7 cells. In addition, 11n caused sub-G1 phase arrest in the cell cycle arrest. Besides, this compound induced anti-angiogenesis in CAM assay and increased the level of caspase-3 by 5.2 fold. The western-blot analysis of the most active compound, 11n, revealed the inhibition of VEGFR-2 phosphorylation. Molecular docking study also showed the important interactions for compound 11n.

Antiproliferative activity. Two series of new tetrahydropyridothienopyrimidines were evaluated for their cytotoxicity against a panel of cell lines, including MCF-7 (breast cancer), SW480 (colon cancer), PC-3 (prostate cancer), HEPG-2 (liver cancer) and HUVEC (human umbilical vein endothelial cell) using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 26 . Sorafenib was used as a positive control. The results are depicted in Table 1. The cytotoxicity is expressed as the concentration that inhibits 50% of cell viability (IC 50 ). As illustrated in Table 1, in general, the target compounds exhibited good cytotoxic activity. The preliminary SAR is evaluated on the basis of cytotoxicity results of phenyl derivatives (11a-k) and pyridine derivatives (11l-11y).The mesomeric effect didn't improve the cytotoxicity of compound 11d in the first series. Those derivatives with methyl and fluorine substituents at meta position are more potent against HUVEC than para-substituted counterparts (11b vs 11c/11e vs 11f/11q vs 11r). The presence of fluorine at para position of the phenyl ring significantly decreases the activity and the change of this atom with chlorine didn't improve the activity. The extra chlorine atom at meta position generated compound 11h which shows a significant inhibitory effect on HUVEC (IC 50 = 7.44 µM). Compounds 11b and 11h are more active against MCF-7 in comparison to other derivatives. In the first series, this electronegative atom at para position, 11f, showed weaker activity against SW480 and HUVEC compared to chlorine-substituted analogue, 11g.
In the second series, against the MCF-7 cell line, compounds from pyridine series, including 11m, 11n, 11p, 11r, 11s, 11w exhibit better anticancer activity than that of sorafenib. Among them, Compound 11n is the most active compound against four cell lines with IC 50 s values of 2.67, 6.84, 7.20 and 2.09 µM in MCF-7, SW-480, HEPG-2, and HUVEC cell lines, respectively. The electronic displacement of compounds 11l, 11n and even the presence of methoxy with positive mesomeric effect resulted in similar activity compared to sorafenib against HUVEC. The activity of these compounds against MCF-7 is stronger than positive control. In this series, comparing the inductive effect of halogens revealed that the most electronegative atom, fluorine, at para position, 11r, resulted in less active compound compared to para chloro substituted compound, 11u, except against MCF-7. The movement of methoxy from meta to para enhances the cytotoxicity of compound 11p against all cell lines except PC-3. The preferred substituent on the phenyl moiety is methyl as the lipophilic group at the meta and para position. Compound 11l exhibits the highest inhibitory activity against four out of five cell lines, including MCF-7, HEPG-2, SW480 and HUVEC. The introduction of chlorine to meta position, 11w, led to the decreased activity compared to 11n except the activity against PC-3. The movement of chlorine to para position (11t vs 11u) increases almost 30 folds the cytotoxicity against HUVEC. Compound 11 × with CF 3 group at meta position shows a lower but not negligible cytotoxicity. From this initial screen, we selected compounds 11l, 11m, 11n from pyridine series and 11b, 11h from phenyl series to investigate the mechanism of activity against MCF-7 and HUVEC cells. The cytotoxicity of compounds 11b, 11h, 11l, 11m, 11n against the normal cell line (MRC5) were also determined ( Table 2). The compound 11n shows IC 50 value of 38.1 μM and the highest selectivity index of 14. Selectivity against MCF-7 versus MRC5 range is between 3.9-14. Results indicated that these compounds have acceptable SI values and expected to be safe.
Apoptosis-inducing activity. The programmed cell death, apoptosis is essential in important biological processes. It is worth mentioning that disease like cancer is considered as a result of disruption in this process. www.nature.com/scientificreports/ The ability of compounds 11b, 11h, 11l, 11m, and 11n at IC 50 concentration in inducing apoptosis on MCF-7, HUVEC cell lines was evaluated 27 . The experiment was conducted by flow cytometry analysis of pigmented cells with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (annexine V-FITC). DMSO and sorafenib were used as the negative and positive control, respectively. The results are depicted in Fig. 2 showing the percentages of necrotic, apoptotic and live cells. The higher percentage of total apoptosis in HUVEC cells compared to MCF-7 is observed for compound 11n. The percentage of early apoptosis for 11l, 11m, 11n are 5.51%, 10.1%, 16.5% and 89.0%, 52.5%, 79.8% of late apoptosis in HUVEC cells. These numbers in MCF-7 cell line are 4.77%, 2.94%, 58.2% and 85.6%, 83.0% and 22.8% for late and early apoptosis, respectively. The apoptosis induced by compound 11n is much greater than that of sorafenib. While in 11a-k series, both selected compounds, 11b, 11h, are unable to induce apoptosis and cells remained live in HUVEC and MCF-7 (The figure is inserted in supporting file). The obtained results and apoptotic cell population confirmed that 11l, 11m, 11n show significant activities in this order 11n > 11l > 11m against HUVEC cell line while against MCF-7 the order is reversed 11m > 11l > 11n. www.nature.com/scientificreports/ The effect of compound 11n on the Caspase-3 level. The activity of caspase (cysteinyl-aspartatespecific proteases) as intracellular cell death enzymes has been characterized. So, the inhibition of caspase-3 is considered enough to block or even slow down the process [28][29][30][31] . By using ELISA assay kit, the bioluminescent intensity of caspase-3 was assessed time-dependently in MCF-7 cells treated with compound 11n at 2.67 μM for 24 h. As shown in Table 3, the treatment of MCF-7 cells with this compound boosted the caspase-3 level by 5.2-fold compared to sorafenib.

Cell cycle arrest.
To further understand the antitumor mechanism of compounds, the impact of the most active compounds on cell cycle progression against HUVEC and MCF-7 were examined at IC 50 concentration by Annexin V-FITC/PI (Propidium iodide) dual staining assay (The figures of 11b, 11 h are inserted in Supporting file) 28 . As shown in Fig. 3, the analysis of cell cycle revealed that compound 11 l increases % the cells in G0 phase to 97.6%. It was observed that compound 11n demonstrates elevation in the cell population of the examined cell www.nature.com/scientificreports/ line, MCF-7, in G0 phase and arrests the cell growth in G0 phase. The percentage of G0 cell is increased to 54.3% and 65.5% in MCF-7 and HUVEC cell lines, respectively. Whereas non-treated cells and sorafenib show 2.0 and 2.2% in this phase. We can conclude that compound 11n induces cellular apoptosis in G0 Phase. An increase in sub-G1 area is an indicative of apoptosis. Compounds 11m, 11l increase the percentage of cell population in the S phase of the cell cycle to 32.9% and 49.8%. These results suggest that they could induce cancer cell cycle arrest (MCF-7) at the S phase.
Anti-angiogenic activity. Since VEGF has been shown to be a strong cell proliferation, and migration inducer, angiogenesis inhibition via VEGF-mediated signaling has been considered as a treatment approach. The hydrogen bonding between NH groups of urea and target proteins were known to give rise to better inhibition of an important growth factor receptor (VEGFR-2) involved in the process of angiogenesis. Thus, the chick chorioallantoic membrane (CAM) assay was utilized as an in-vivo technique to evaluate the anti-vascular effects. Therefore, the anti-angiogenic strength of compounds were investigated by a cheap and simple CAM assay (Figs. 4, 5) 32,33 . The anti-angiogenic strength was measured and compared with DMSO (control) and Sorafenib as a positive control. Compounds 11h, 11m, 11n exhibit slightly weaker inhibitory effects on growing CAM compared to the positive control.

Molecular docking study.
To understand the structure-activity relationship, the compound 11n was docked into the catalytic site of VEGFR-2 protein (PDB code: 3WZE) 34  Western blot analysis. Western blot was performed to investigate the effect of the most potent compound 11n on VEGFR-2 protein's phosphorylation in VEGFR-2 overexpressed HUVECs cell membrane 35 . It was is observed that, after 48 and 72 h, the phosphorylation of VEGFR-2 decreases compared to the positive control ( Fig. 7).

Conclusion.
In summary, we have successfully synthesized a series of tetrahydropyridothienopyrimdines with antiproliferative and anti-angiogenic properties. Among the synthesized compounds, we found that compound 11n shows good cytotoxic activities Cellular mechanistic studies of compound 11n disclosed that it prompts apoptosis, cell growth cessation at G0 phase and it reinforces apoptosis via activation of caspase-3. The anti-angiogenic strength and western-blot analysis were performed to investigate the mechanism of action. Hydrogen bonding with urea and pyrimidine part with key amino acids and π-stacking interactions of thiadiazole with Phe 1047 showed that compound 11n with proper binding modes can be probably used as principle templates for further investigations. Mechanistic studies and low toxicity profile showed that this core structure has the potential value as a promising candidate for further modification to find novel therapeutic agents.

Methods
Chemistry. All chemicals were prepared from Merck and Sigma. Nicolet FT-IR Magna 550 spectrometer was used to record Infrared spectra. The Bruker FT-500, 300 MHz spectrometers using DMSO-d 6 , CDCl 3 and TMS as the internal standard were used to capture 1 H NMR spectra and 13 C NMR.

Synthesis of 1-substituted piperidin-4-one (3).
A mixture of piperidin-4-one hydrochloride 1 (15 mmol) with K 2 CO 3 (20 mmol) in DMF (20 mL) was stirred at 80 °C. After 30 min, 4-(chloromethyl)pyridine or benzylchloride 2 (15 mmol) was added to the mixture which was heated for further 5 h. Upon completion, the mixture was cooled to the ambient temperature, water (30 mL) was added and extracted with EtOAc (3 × 45 mL and brine and the solvent was removed under the reduced pressure. The residue was purified by column chromatography using n-hexane/EtOAc (6:1) as eluent to afford compound 3 as yellow oil in 70% yield.  and morpholine (30 mmol) was heated in EtOH (20 mL) under reflux conditions for 30 min. Then, sulfur (18 mmol) was added gradually over 5 min. The resulting mixture was refluxed for further 3 h. After the consumption of starting materials according to the TLC analysis, the reaction was cooled to the ambient temperature and stirred overnight. The precipitated product was filtered and washed with diethyl ether (5 mL) to afford pure compound 4 as a yellow solid.

Synthesis of 5-amino-1,3,4-thiadiazole-2-thiol (8).
To the mixture of thiosemicarbazide 7 (15 mmol) and Na 2 CO 3 (15 mmol) in absolute ethanol (15 mL), carbon disulfide (30 mmol) in EtOH (5 mL) was added dropwise and the obtained mixture was heated under reflux conditions for 8 h. As the completion of reaction was confirmed by TLC, the solvent was removed under reduced pressure. The residue was diluted through the addition of water (50 mL), then, concentrated HCl solution was added dropwise to precipitate compound 8 as yellow solid in 80% yield.

Apoptosis inducing analysis.
For determination of apoptosis induction, the best concentration (IC 50 ) of the most active compounds were selected and evaluated on HUVEC, MCF-7 cell lines. After incubation of in a 6-well plate at 37 °C for 24 h, they were treated with selected compounds at their IC 50 concentration for 48 h. Then, cells were trypsinized, rinsed with phosphate-bufferede buffered saline (PBS) and then centrifuged at 1200 rpm for 3 min. After this, the binding buffer (500 μL) was added to the resulting cells, followed by addition and mixing with Annexin V-APC and PI (5 μL). After this, the samples were incubated in dark for 10 to 15 min at room temperature, and then the cellular analysis was measured by flow cytometer (FACS Calibur Bectone-Dickinson). www.nature.com/scientificreports/

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.